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bioRxiv · 10.64898/2026.09.03.749169

Molecular Determinants of Sequence-Dependent N-glycosylation by the Human Oligosaccharyltransferase

Andjelkovic, M.·Belen-Aguilar, J. F.·Belda, D.·Ruiz-Pernia, J. J.·Tunon, I.·Mingarro, I.
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Abstract

Protein N-glycosylation is catalysed by the oligosaccharyltransferase (OST) complex, yet how local sequon composition controls substrate selection by human OST remains poorly understood. Here we combine atomistic molecular dynamics, alchemical free-energy calculations, in cellulo glycosylation assays and human glycoproteome analysis to define the molecular basis of sequence-dependent N-glycosylation. We built, to our knowledge, the first atomistic model of human OST with both the lipid-linked oligosaccharide donor and an acceptor peptide simultaneously accommodated in a catalytically competent complex. Simulations show that conserved STT3 motifs stabilize the acceptor Asn and explain how substitutions at positions 0, +1 and +2 alter binding and productive geometry. Cellular assays in HEK 293T cells validate the predicted loss of glycosylation for N0Q sequon, +1 Pro and the +2 hierarchy Thr > Ser >> Cys. Analysis of 14,800 human glycosites confirms these rules at proteome scale.

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Andjelkovic, M., Belen-Aguilar, J. F., Belda, D., Ruiz-Pernia, J. J., Tunon, I., Mingarro, I.. 2026-09-07. Molecular Determinants of Sequence-Dependent N-glycosylation by the Human Oligosaccharyltransferase. https://doi.org/10.64898/2026.09.03.749169

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